Aryl/Alkyl Succinic Anhydride Hyaluronan Derivatives

ABSTRACT

The present invention relates to the modification of hyaluronic acid (HA) with aryl/alkyl succinic anhydrides (ASA) to produce aryl/alkyl succinic anhydride HA derivatives, to the derivatives as such, and to their applications and uses, particularly in the cosmetic and biomedical industries. The ASA-HA derivatives are expected to have interesting properties that can be used for advanced formulation (bind stronger to the skin compared to non-modified HA), possibly also in delivery systems for actives or drugs by encapsulation (nano/micro capsules) or formation of nano/micro spheres. Further, the low MW ASA-HA derivatives are expected to penetrate the skin more efficiently than non-modified HA of the same MW.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/554,190 filed on Jul. 20, 2012, which is a continuation of U.S.patent application Ser. No. 13/164,012 filed on Jun. 20, 2011, nowabandoned, which is a divisional of U.S. patent application Ser. No.11/572,954, now U.S. Pat. No. 7,993,678, which is a 35 U.S.C. 371national application of PCT/DK2006/000523 filed on Sep. 26, 2006, whichclaims priority or the benefit under 35 U.S.C. 119 of Danish applicationno. PA 2005 01332 filed on Sep. 26, 2005 and U.S. provisionalapplication No. 60/721,232 filed on Sep. 27, 2005. The contents of theseapplications are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the modification of hyaluronic acid(HA) with aryl- or alkyl succinic anhydride (ASA) to produce aryl/alkylsuccinic anhydride HA derivatives (ASA-HA), to the ASA-HA derivatives assuch, and to their applications and uses, particularly in the cosmeticsand biomedical industries.

BACKGROUND OF THE INVENTION

The most abundant heteropolysaccharides of the body are theglycosaminoglycans. Glycosaminoglycans are unbranched carbohydratepolymers, consisting of repeating disaccharide units (only keratansulphate is branched in the core region of the carbohydrate). Thedisaccharide units generally comprise, as a first saccharide unit, oneof two modified sugars—N-acetylgalactosamine (GalNAc) orN-acetylglucosamine (GlcNAc). The second unit is usually an uronic acid,such as glucuronic acid (GlcUA) or iduronate.

Glycosaminoglycans are negatively charged molecules, and have anextended conformation that imparts high viscosity when in solution.Glycosaminoglycans are located primarily on the surface of cells or inthe extracellular matrix. Glycosaminoglycans also have lowcompressibility in solution and, as a result, are ideal as aphysiological lubricating fluid, e.g., joints. The rigidity ofglycosaminoglycans provides structural integrity to cells and providespassageways between cells, allowing for cell migration. Theglycosaminoglycans of highest physiological importance are hyaluronan,chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate, andkeratan sulfate. Most glycosaminoglycans bind covalently to aproteoglycan core protein through specific oligosaccharide structures.Hyaluronan forms large aggregates with certain proteoglycans, but is anexception as free carbohydrate chains form non-covalent complexes withproteoglycans.

Numerous roles of hyaluronan in the body have been identified (see,Laurent and Fraser, 1992, FASEB J. 6: 2397-2404; and Toole B. P., 1991,“Proteoglycans and hyaluronan in morphogenesis and differentiation.” In:Cell Biology of the Extracellular Matrix, pp. 305-341, Hay E. D., ed.,Plenum, New York). Hyaluronan is present in hyaline cartilage, synovialjoint fluid, and skin tissue, both dermis and epidermis. Hyaluronan isalso suspected of having a role in numerous physiological functions,such as adhesion, development, cell motility, cancer, angiogenesis, andwound healing. Due to the unique physical and biological properties ofhyaluronan, it is employed in eye and joint surgery and is beingevaluated in other medical procedures.

The terms “hyaluronan” or “hyaluronic acid” are used in literature tomean acidic polysaccharides with different molecular weights constitutedby residues of D-glucuronic and N-acetyl-D-glucosamine acids, whichoccur naturally in cell surfaces, in the basic extracellular substancesof the connective tissue of vertebrates, in the synovial fluid of thejoints, in the endobulbar fluid of the eye, in human umbilical cordtissue and in cocks' combs.

The term “hyaluronic acid” is in fact usually used as meaning a wholeseries of polysaccharides with alternating residues of D-glucuronic andN-acetyl-D-glucosamine acids with varying molecular weights or even thedegraded fractions of the same, and it would therefore seem more correctto use the plural term of “hyaluronic acids”. The singular term will,however, be used all the same in this description; in addition, theabbreviation “HA” will frequently be used in place of this collectiveterm.

HA plays an important role in the biological organism, as a mechanicalsupport for the cells of many tissues, such as the skin, tendons,muscles and cartilage, it is a main component of the intercellularmatrix. HA also plays other important parts in the biological processes,such as the moistening of tissues, and lubrication.

HA may be extracted from the above mentioned natural tissues, althoughtoday it is preferred to prepare it by microbiological methods tominimize the potential risk of transferring infectious agents, and toincrease product uniformity, quality and availability.

HA and its various molecular size fractions and the respective saltsthereof have been used as medicaments, especially in treatment ofarthropathies, as an auxiliary and/or substitute agent for naturalorgans and tissues, especially in ophthalmology and cosmetic surgery,and as agents in cosmetic preparations. Products of hyaluronan have alsobeen developed for use in orthopaedics, rheumatology, and dermatology.

HA may also be used as an additive for various polymeric materials usedfor sanitary and surgical articles, such as polyurethanes, polyestersetc. with the effect of rendering these materials biocompatible.

The ASA modification or derivatization is well established in the paperindustry where alkyl succinic anhydrides have been used to make papersurfaces (cellulosic) more water resistant (Chen and Woodward, August1986, Optimizing the emulsification and sizing of alkenyl succinicanhydride, Tappi Journal 95-97). In the food industry2-octen-1-ylsuccinic anhydride (OSA) modified starches have been used tostabilize oil/water emulsions, e.g., low fat margarines and mayonnaises,(Jarowenko, W. (In: Properties and uses of modified starches, 1986, Ed.:O. Wurzburg) Acetylated starch and miscellaneous organic esters, pp55-77). Further, the rheological properties of OSA modified starches arevery different compared to non-modified starches (Park, Chung, and Yoo,2004, Effects of octenylsuccinylation on rheological properties of cornstarch pastes, Starch 56: 399-406).

The advantages of the ASA derivatization procedure are, e.g., that theproducts are non-toxic, the chemicals cheap, and the reaction is aone-step procedure (Trubiano, PC. [In: Properties and uses of modifiedstarches, 1986, Ed.: O. Wurzburg] Succinate and substituted succinatederivatives of starch, pp 131-147; Wurzburg, O B. 1995. Modifiedstarches, In: Food Science and Technology, Vol. 67, New York, pp.67-97).

According to earlier studies on starches, both primary and secondaryhydroxyl groups react with OSA (Shogren, Viswanathan, Felker, and Gross,2000, Distribution of octenyl succinate groups in octenyl succinicanhydride modified waxy maize starch, Starch 52:196-204).

SUMMARY OF THE INVENTION

There is a need, particularly in the cosmetics and biomedicalindustries, for hyaluronic acid based compounds or derivatives that havecertain altered characteristics as compared to non-modified HA.Properties of interest are the improved ability to stabilize foam, andthe ability to blend with non-hydrophilic materials, such as is usedtypically in cosmetics products.

The invention provides amphiphilic HA-derivative products withproperties of benefit in cosmetics or biomedical applications. Theseproducts bind more strongly to the skin so that they are not so easilywashed of. The ASA-HA derivatives are also suitable for use in moreadvanced cosmetic or biomedical formulations, e.g. in the formation ofnano/macro capsules or nano/macro spheres for delivery of activecompounds or drugs. ASA-HA derivatives of lower molecular weight (MW)will penetrate the skin more efficiently than non-derivatized HA ofcomparable MW.

In the examples herein, hyaluronic acid (HA) was modified withalkyl/aryl succinic anhydrides (ASA) under alkaline conditions (pH>8.0)in water. The resulting products were purified (precipitation ordialysis). These purified products formed partially water-insolubleaggregates in water. A 1% solution was showed to stabilize foam (reducedsurface tension+increased interfacial viscosity). ¹H NMR spectroscopyconfirmed that the chemical structure of the HA “backbone” in theresulting product was unchanged, except for the introduction of ASAhalf-ester groups up to a degree of substitution (DS) of about 18%.

Accordingly, in a first aspect, the invention relates to a hyaluronicacid derivative comprising ‘n’ repeating units and having the generalstructural formula (I) at pH 8-9:

wherein in at least one repeating unit one or more of R1, R2, R3, R4comprise an ester bound alkyl/aryl-succinic acid having the generalstructural formula (II) at pH 8-9, and otherwise R1, R2, R3, R4 arehydroxyl groups, OH:

wherein at least one of R5, R6, R7, R8 comprises an alkyl- oraryl-group, and otherwise R5, R6, R7, R8 are hydrogen atoms, H, andwherein the Oxygen labelled “ester” partakes the ester bond withstructure (I).

In other words, an aspect of the invention relates to a hyaluronic acidderivative, wherein one or more hydroxyl-groups of the hyaluronic acidhave been substituted in a reaction with one or more alkyl/aryl-succinicanhydrides (ASA), to form an ester-bond between the hyaluronic acid andthe resulting one or more alkyl/aryl-succinic acids.

In a second aspect, the invention relates to a process of producing ahyaluronic acid derivative, comprising the steps of:

(a) reacting a hyaluronic acid (HA) with one or more alkyl/aryl-succinicanhydrides (ASA) having the general structural formula shown in (III)

under alkaline conditions in an aqueous solution, whereby the hyaluronicacid derivative is formed; and

(b) recovering the hyaluronic acid derivative.

In a third aspect, the invention relates to a composition comprising ahyaluronic acid derivative as defined in the first aspect and an activeingredient, preferably the active ingredient is a pharmacologicallyactive agent.

A fourth aspect of the invention relates to a pharmaceutical compositioncomprising an effective amount of a hyaluronic acid derivative asdefined in the first aspect, together with a pharmaceutically acceptablecarrier, excipient or diluent.

A fifth aspect relates to a pharmaceutical composition comprising aneffective amount of a hyaluronic acid derivative as defined in the firstaspect as a vehicle, together with a pharmacologically active agent.

A sixth aspect relates to a cosmetic article comprising as an activeingredient an effective amount of a hyaluronic acid derivative asdefined in the first aspect or a composition as defined in any of thesecond, third, or fourth aspects.

In a seventh aspect, the invention relates to a sanitary, medical orsurgical article comprising a hyaluronic acid derivative as defined inthe first aspect or a composition as defined in any of the second,third, or fourth aspects, preferably the article is a diaper, a sanitarytowel, a surgical sponge, a wound healing sponge, or a part comprised ina band aid or other wound dressing material.

An important aspect relates to a medicament capsule, microcapsule,nanocapsules, microsphere or nanosphere comprising a hyaluronic acidderivative as defined in the first aspect or a composition as defined inany of the third, fourth, or fifth aspects.

Final aspects of the invention relate to methods of performingprocedures in ophthalmology, in the treatment of osteoarthritis orcancer, hair loss or baldness, of treating a wound, of performing dermalor transdermal administration of a pharmacologically active agent, ordermal administration of a cosmetic, the improvement which comprises theuse of a hyaluronic acid derivative as defined in the first aspect, or acomposition as defined in any of the second, third, or fourth aspects.

A number of aspects relate to uses of a hyaluronic acid derivative asdefined in any of the first aspects or a composition as defined in anyof the third, fourth, or fifth aspects for the manufacture of amedicament for the treatment of osteoarthritis, cancer, the manufactureof a medicament for an ophthalmic treatment, the manufacture of amedicament for the treatment of a wound, the manufacture of a medicamentfor angiogenesis, the manufacture of a medicament for the treatment ofhair loss or baldness, or the manufacture of a moisturizer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. The schematic ASA modification of HA is illustrated in FIG. 1.

FIG. 2. The partially assigned ¹H NMR spectrum of the 100 kDa OSA-HA(14919-033) of examples 5 and 6.

FIG. 3. The chemical structure of the ASAs used to modify HA.

FIG. 4. Haug's triangle summarizes the relationship between Rg and Mwfor different polymer conformations. By plotting radius of gyration (Rg)as against molecular weight (Mw) in double logarithmic scale, one canobtain information about the conformation of the polymer.

FIG. 5. Concentration profiles (RI) for the ASA modified LMW HA ofexample 12 below.

FIG. 6. Results of surface tension measurements on LMW OSA-HA usingsurface tensiometer (Wilhemy plate) as described in example 13.

FIG. 7. Shows the emulsification properties of ASA-HA after 24 hours and8 weeks with ethylhexyl palmitate (cosmetic oil) prepared as describedin example 15.

FIG. 8. The critical aggregation concentration (CAC) of an OSA-HAderivative was determined by calorimetry as described in Example 18,enthalpy variations (ΔH) in the sample cell were recorded over time asshown in FIG. 8.

FIG. 9. Enthalpy variation (ΔH) of Example 18 was plotted as a functionof the OSA-HA concentration in the sample cell, and the CAC of OSA-HAwas determined at the break of the curve. Each experiment was repeatedthree times and the CAC was provided as an averaged value. For example,the CAC of OSA-HA with a degree of substitution (DS) of 16% was 0.45mg/mL.

FIG. 10. Shows the wavelength of maximum emission as a function ofOSA-HA (DS=44%) concentration in buffer 1 as described in Example 19.

FIG. 11. Shows the wavelength of maximum emission as a function ofOSA-HA (DS=44%) concentration, and that the CAC decreases as theconcentration of NaCl increases, as described in Example 20.

FIG. 12. Shows the Zeta potential of OSA-HA (DS=44%, 1 mg/mL) in 10-3 MNaCl as determined in Example 21.

FIG. 13. Shows a transmission electron micrograph of OSA-HA (DS=44%)polymeric micelles from Example 22, that are spherical in shape and havesubmicronic dimensions typically from 50 to 200 nm.

DETAILED DESCRIPTION OF THE INVENTION Hyaluronic Acid

“Hyaluronic acid” is defined herein as an unsulphated glycosaminoglycancomposed of repeating disaccharide units of N-acetylglucosamine (GlcNAc)and glucuronic acid (GlcUA) linked together by alternating beta-1,4 andbeta-1,3 glycosidic bonds. Hyaluronic acid is also known as hyaluronan,hyaluronate, or HA. The terms hyaluronan and hyaluronic acid are usedinterchangeably herein.

Rooster combs are a significant commercial source for hyaluronan.Microorganisms are an alternative source. U.S. Pat. No. 4,801,539discloses a fermentation method for preparing hyaluronic acid involvinga strain of Streptococcus zooepidemicus with reported yields of about3.6 g of hyaluronic acid per liter. European Patent No. EP 0694616discloses fermentation processes using an improved strain ofStreptococcus zooepidemicus with reported yields of about 3.5 g ofhyaluronic acid per liter. As disclosed in WO 03/054163 (Novozymes),which is incorporated herein in its entirety, hyaluronic acid or saltsthereof may be recombinantly produced, e.g., in a Gram-positive Bacillushost.

Hyaluronan synthases have been described from vertebrates, bacterialpathogens, and algal viruses (DeAngelis, 1999, Cell. Mol. Life Sci. 56:670-682). WO 99/23227 discloses a Group I hyaluronate synthase fromStreptococcus equisimilis. WO 99/51265 and WO 00/27437 describe a GroupII hyaluronate synthase from Pasturella multocida. Ferretti et al.disclose the hyaluronan synthase operon of Streptococcus pyogenes, whichis composed of three genes, hasA, hasB, and hasC, that encodehyaluronate synthase, UDP glucose dehydrogenase, and UDP-glucosepyrophosphorylase, respectively (Proc. Natl. Acad. Sci. USA. 98:4658-4663 (2001)). WO 99/51265 describes a nucleic acid segment having acoding region for a Streptococcus equisimilis hyaluronan synthase.

Since the hyaluronan of a recombinant Bacillus cell is expresseddirectly to the culture medium, a simple process may be used to isolatethe hyaluronan from the culture medium. First, the Bacillus cells andcellular debris are physically removed from the culture medium. Theculture medium may be diluted first, if desired, to reduce the viscosityof the medium. Many methods are known to those skilled in the art forremoving cells from culture medium, such as centrifugation ormicrofiltration. If desired, the remaining supernatant may then befiltered, such as by ultrafiltration, to concentrate and remove smallmolecule contaminants from the hyaluronan. Following removal of thecells and cellular debris, a simple precipitation of the hyaluronan fromthe medium is performed by known mechanisms. Salt, alcohol, orcombinations of salt and alcohol may be used to precipitate thehyaluronan from the filtrate. Once reduced to a precipitate, thehyaluronan can be easily isolated from the solution by physical means.The hyaluronan may be dried or concentrated from the filtrate solutionby using evaporative techniques known to the art, such as spray drying.

The first aspect of the invention relates to a hyaluronic acidderivative comprising n repeating units and having the formula (I) at pH8-9:

wherein in at least one repeating unit one or more of R1, R2, R3, R4comprise an ester bound alkyl/aryl-succinic acid having the generalstructural formula (II) at pH 8-9, and otherwise R1, R2, R3, R4 arehydroxyl groups, OH:

wherein at least one of R5, R6, R7, R8 comprises an alkyl- oraryl-group, and otherwise R5, R6, R7, R8 are hydrogen atoms, H, andwherein the Oxygen labelled “ester” partakes the ester bond withstructure (I).

In a preferred embodiment of the first aspect, two or more of R1, R2,R3, R4 comprise one or more ester bound alkyl/aryl-succinic acids havingthe general structural formula (II) at pH 8-9; preferably three or moreof R1, R2, R3, R4 comprise one or more ester bound alkyl/aryl-succinicacids having the general structural formula (II) at pH 8-9.

In another preferred embodiment of the first aspect, at least one of R5,R6, R7, R8 comprises an alkyl-group, preferably at least two of R5, R6,R7, R8 comprise an alkyl-group, more preferably at least three of R5,R6, R7, R8 comprise an alkyl-group; preferably the alkyl-group comprisesa C₁-C₂₀ alkyl group, preferably propyl, 2-octenyl, 2-nonenyl,2-dodecenyl, 2-hexadecenyl, or 2-octadecenyl.

Yet another preferred embodiment relates to the HA derivative of thefirst aspect, wherein at least one of R5, R6, R7, R8 comprises anaryl-group, preferably at least two of R5, R6, R7, R8 comprise anaryl-group, more preferably at least three of R5, R6, R7, R8 comprise anaryl-group; and preferably the aryl-group is phenyl.

It is preferred that R5, R6, R7, R8 comprise two or more differentalkyl- and/or aryl-groups, preferably chosen from propyl, 2-octenyl,2-nonenyl, 2-dodecenyl, 2-hexadecenyl, 2-octadecenyl, and phenyl.

Molecular Weight

The level of hyaluronic acid may be determined according to the modifiedcarbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334).Moreover, the average molecular weight of the hyaluronic acid may bedetermined using standard methods in the art, such as those described byUeno et al., 1988, Chem. Pharm. Bull. 36: 4971-4975; Wyatt, 1993, Anal.Chim. Acta 272: 1-40; and Wyatt Technologies, 1999, “Light ScatteringUniversity DAWN Course Manual” and “DAWN EOS Manual” Wyatt TechnologyCorporation, Santa Barbara, Calif.

In a preferred embodiment, the hyaluronic acid derivatives obtained bythe methods of the present invention has a molecular weight of about 800to about 10,000,000 Da. In a more preferred embodiment, the hyaluronicacid derivatives obtained by the methods of the present invention has amolecular weight of about 1,000 to about 9,000,000 Da; about 2,000 toabout 10,000,000 Da; about 4,000 to about 10,000,000 Da; about 8,000 toabout 10,000,000 Da; about 10,000 to about 10,000,000 Da; or about25,000 to about 5,000,000 Da. In an even more preferred embodiment, thehyaluronic acid derivatives obtained by the methods of the presentinvention has a molecular weight of about 50,000 to about 3,000,000 Da.

Another preferred embodiment relates to the product of the first aspect,wherein the hyaluronic acid or salt thereof has a molecular weight inthe range of between 300,000 and 3,000,000; preferably in the range ofbetween 400,000 and 2,500,000; more preferably in the range of between500,000 and 2,000,000; and most preferably in the range of between600,000 and 1,800,000 Da.

Where recombinantly produced hyaluronic acid or salt thereof is used inthe methods of the invention to manufacture the products or compositionsof the invention, it may be advantageous for some applications to firstreduce the average molecular weight of the hyaluronic acid or derivativeor salts thereof. For instance, it has been stated by severalmanufacturers of so-called low-molecular weight fractions of hyaluronicacid, that it is capable of penetrating the skin barrier to reestablishthe natural content of hyaluronic acid in the skin, therefore suchfractions are particularly suitable for cosmetic compositions sold asanti-skin-ageing and anti-wrinkle agents. For food applications, low MWhyaluronic acid has been shown to penetrate the gastrointestinalbarrier, thereby increasing its bioavailability. Finally, low MWhyaluronic acid exhibits anti-inflammatory effect and have potentialapplications in the treatment of inflammatory diseases. A reduction ofthe average molecular weight of a hyaluronic acid or derivative or saltthereof may be achieved by standard methods in the art, such as, simpleheat treatment, enzymatic degradation, ultrasound sonication, or acidhydrolysis. See, e.g., U.S. Pat. No. 6,020,484, which describes anultrasonication technique of HA including NaOCp as additive, andMiyazaki et al., 2001, Polymer Degradation and Stability 74: 77-85.

Accordingly, a preferred embodiment relates to the HA derivative of theinvention, wherein the hyaluronic acid or derivative or salt thereof hasa low average molecular weight in the range of between 800 and10,000,000 Da; preferably in the range of between 10,000 and 1,500,000Da; preferably in the range of between 10,000 and 50,000 Da; orpreferably in the range of between 50,000 and 500,000 Da; even morepreferably in the range of between 80,000 and 300,000 Da.

Degree of Substitution (DS)

DS was determined by ¹H NMR spectroscopy (10 mg/ml, D₂O, 80° C., 128scans, 400 MHz) according to example 6 below, wherein the peaks from theOSA group were assigned by use of a 2D-NMR (gCOSY). The DS was thencalculated by comparing the intensity of the vinyl protons of OSA (5.4and 5.6 ppm) with that of the acetyl protons (2.0 ppm).

In a preferred embodiment the HA derivative of the first aspect has aDegree of Substitution (DS) in the range of 0.1-100%, preferably 1-90%,more preferably 2-80%, still more preferably 4-70%, even more preferably8-60%, or 10-50%, 14-40%, 16-30%, or most preferably in the range of18-25%.

Alkyl/Aryl-Succinic Anhydride (ASA)

In a preferred embodiment of the invention, the one or morealkyl/aryl-succinic anhydrides (ASA) have the general structural formula(III):

In one preferred embodiment, at least one of R5, R6, R7, R8 comprises analkyl-group, more preferably at least two of R5, R6, R7, R8 comprise analkyl-group, even more preferably at least three of R5, R6, R7, R8comprise an alkyl-group; and preferably the alkyl-group comprises aC₁-C₂₀ alkyl group, preferably propyl, 2-octenyl, 2-nonenyl,2-dodecenyl, 2-hexadecenyl, or 2-octadecenyl.

In another preferred embodiment, at least one of R5, R6, R7, R8comprises an aryl-group, preferably at least two of R5, R6, R7, R8comprise an aryl-group, more preferably at least three of R5, R6, R7, R8comprise an aryl-group, which preferably comprises phenyl.

In yet another preferred embodiment R5, R6, R7, R8 comprises two or moredifferent alkyl- and/or aryl-groups, preferably chosen from propyl,2-octenyl, 2-nonenyl, 2-dodecenyl, 2-hexadecenyl, 2-octadecenyl, andphenyl.

In still another preferred embodiment, the one or more ASA comprise anyof the structural formulae shown in FIG. 3.

Production

In the methods of the present invention recombinantly produced HA may beused that is produced by a process, wherein the HA-producing host cellsare cultivated in a nutrient medium suitable for production of thehyaluronic acid using methods known in the art. For example, the cellmay be cultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the enzymes involved inhyaluronic acid synthesis to be expressed and the hyaluronic acid to beisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). The secreted hyaluronic acid can be recovered directly fromthe medium.

The resulting hyaluronic acid may be isolated by methods known in theart. For example, the hyaluronic acid may be isolated from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. The isolated hyaluronic acid may then be further purifiedby a variety of procedures known in the art including, but not limitedto, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

Host Cells

A preferred embodiment relates to where the hyaluronic acid or saltthereof is recombinantly produced, preferably by a Gram-positivebacterium or host cell, more preferably by a bacterium of the genusBacillus.

The host cell may be any Bacillus cell suitable for recombinantproduction of hyaluronic acid. The Bacillus host cell may be a wild-typeBacillus cell or a mutant thereof. Bacillus cells useful in the practiceof the present invention include, but are not limited to, Bacillusagaraderhens, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacilluscoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.Mutant Bacillus subtilis cells particularly adapted for recombinantexpression are described in WO 98/22598. Non-encapsulating Bacilluscells are particularly useful in the present invention.

In a preferred embodiment, the Bacillus host cell is a Bacillusamyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacilluslicheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. Ina more preferred embodiment, the Bacillus cell is a Bacillusamyloliquefaciens cell. In another more preferred embodiment, theBacillus cell is a Bacillus clausii cell. In another more preferredembodiment, the Bacillus cell is a Bacillus lentus cell. In another morepreferred embodiment, the Bacillus cell is a Bacillus licheniformiscell. In another more preferred embodiment, the Bacillus cell is aBacillus subtilis cell. In a most preferred embodiment, the Bacillushost cell is Bacillus subtilis A164Δ5 (see U.S. Pat. No. 5,891,701) orBacillus subtilis 168Δ4.

Transformation of the Bacillus host cell with a nucleic acid constructof the present invention may, for instance, be effected by protoplasttransformation (see, e.g., Chang and Cohen, 1979, Molecular GeneralGenetics 168: 111-115), by using competent cells (see, e.g., Young andSpizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), byelectroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987,Journal of Bacteriology 169: 5271-5278).

Salts and Crosslinked HA

A preferred embodiment relates to a hyaluronic acid derivative of thefirst aspect, which comprises an inorganic salt of hyaluronic acid,preferably sodium hyaluronate, potassium hyaluronate, ammoniumhyaluronate, calcium hyaluronate, magnesium hyaluronate, zinchyaluronate, or cobalt hyaluronate.

The preparation of a crosslinked HA or salt thereof, which is preparedby crosslinking HA with a polyfunctional epoxy compound is disclosed inEP 0161887 B1. Total or partial crosslinked esters of HA with analiphatic alcohol, and salts of such partial esters with inorganic ororganic bases, are disclosed in U.S. Pat. No. 4,957,744. Other ways ofcross-linking HA are disclosed in U.S. Pat. Nos. 5,616,568, 5,652,347,and 5,874,417.

Crosslinked HA may also be prepared by treating HA with boric acid, asfollows: Dried sodium hyaluronate (Na-HA, 203 mg), recombinantlyproduced in a Bacillus subtilis by fermentation (WO 03/054163;Novozymes), was dissolved into 0.2 M NaOH to give a 4% solution. Boricacid (35 mg (approx. 1 equivalent of HA disaccharide) was added and thesample was stirred at room temperature for 1.5 h, and then stored at 5°C. for approx. 2.5 days. A control sample was prepared in parallelexactly as described above, but without boric acid. The viscosity of theresulting HA-borate hydrogel was measured at 25° C. using a Carrimed CSLcontrolled stress rheometer (cone geometry: 6 cm,)2°. The viscositydepended on the shear rate and increased at least 4-fold (from 4.2- to8.4 fold) in the HA-borate hydrogel as compared to the control sample,indicating formation of a cross-linked network. New peaks at 1200 and945 cm-1 were observed on the FT-IR spectrum of the HA-borate hydrogel,when compared to a standard spectrum of Na-HA, corresponding to thepresence of newly formed borate esters in the crosslinked HA-boratehydrogel.

Accordingly, a preferred embodiment relates to the HA derivative of thefirst aspect, which comprises crosslinked hyaluronic acid or saltthereof, preferably the hyaluronic acid is crosslinked with boric acid,and more preferably the crosslinked hyaluronic acid comprises borateesters.

Particle Size

A preferred HA derivative of the first aspect has a particle size the 50percentile of which, D₅₀, is between 10 and 1,000 microns, preferablybetween 100 and 1,000 microns, more preferably between 150 and 900microns, and even more preferably between 200 and 800 microns, asdetermined by laser diffraction measurement of the particles suspendedin isopropanol.

In a preferred embodiment, the polydispersity of a HA derivative of thefirst aspect is measured as the SPAN value, which is calculatedaccording to the following formula: SPAN=(D₉₀−D₁₀)/D₅₀, and the SPANvalue is between 1.0 and 2.5; preferably the SPAN value is between 1.2and 2.2; more preferably the SPAN value is between 1.5 and 1.9; and mostpreferably the SPAN value is between 1.6 and 1.8.

Microparticles

As shown in the examples below, the present invention provides ASA-HAderivatives that are capable of forming micro- or nanoparticles, ormicro- or nanocapsules. Such particles or capsules, or compositionscomprising these, may of use in a large number of commercial andscientific applications, such as in cosmetics or in generaldrug-delivery.

Other Ingredients

In a preferred embodiment, the compositions comprising a HA derivativeof the invention may also comprise other ingredients, preferably one ormore active ingredients, preferably one or more pharmacologically activesubstances, and also preferably a water-soluble excipient, such aslactose.

Non-limiting examples of an active ingredient or pharmacologicallyactive substance which may be used in the present invention includeprotein and/or peptide drugs, such as, human growth hormone, bovinegrowth hormone, porcine growth hormone, growth homorne releasinghormone/peptide, granulocyte-colony stimulating factor, granulocytemacrophage-colony stimulating factor, macrophage-colony stimulatingfactor, erythropoietin, bone morphogenic protein, interferon orderivative thereof, insulin or derivative thereof, atriopeptin-III,monoclonal antibody, tumor necrosis factor, macrophage activatingfactor, interleukin, tumor degenerating factor, insulin-like growthfactor, epidermal growth factor, tissue plasminogen activator, factorVII, factor VIII, and urokinase.

A water-soluble excipient may be included for the purpose of stabilizingthe active ingredient(s), such excipient may include a protein, e.g.,albumin or gelatin; an amino acid, such as glycine, alanine, glutamicacid, arginine, lysine and a salt thereof; carbohydrate such as glucose,lactose, xylose, galactose, fructose, maltose, saccharose, dextran,mannitol, sorbitol, trehalose and chondroitin sulphate; an inorganicsalt such as phosphate; a surfactant such as TWEEN® (ICI), poly ethyleneglycol, and a mixture thereof. The excipient or stabilizer may be usedin an amount ranging from 0.001 to 99% by weight of the product.

Several aspects of the invention relate to various compositions andpharmaceutical comprising, among other constituents, an effective amountof the product as defined in the first aspect, and an active ingredient,preferably the active ingredient is a pharmacologically active agent; apharmaceutically acceptable carrier, excipient or diluent, preferably awater-soluble excipient, and most preferably lactose.

In addition, aspects of the invention relate to articles comprising a HAderivative as defined in the first aspect or a composition as defined inthe aspects and embodiments above, e.g., a cosmetic article, a sanitaryarticle, a medical or surgical article. In a final aspect the inventionrelates to a medicament capsule or microcapsule comprising a product asdefined in the first aspect or a composition as defined in other aspectsand embodiments of the invention.

EXAMPLES Materials

High molecular weight (High-MW) Hyaluronic acid (HA):

-   -   (batch MAG 30014)    -   (batch MAF 145 SD)

Low molecular weight (Low-MW) HA:

-   -   100 kDa (batch 14919-021)    -   30 kDa (batch 14919-032)    -   23 kDa    -   14 kDa

Alkyl /Aryl succinic anhydrides:

-   -   cis/trans-2-octen-1-ylsuccinic anhydride (OSA), Aldrich Chemical        Company (d.: 1.000, MW 220.27, 97% purity).    -   Phenylsuccinic anhydride (PhSA) (Dry powder, MW 176.17).    -   Nonenylsuccinic anhydride (NSA) Aldrich Chemical Company (d.:        1.032, MW 224.30, 95+% purity, 1JS38, 246-00198-1).    -   Dodecenylsuccinic anhydride (DSA) (d.: 1.01, MW 266.38, 1JS38,        246-00168).    -   Tetrapropylsuccinic anhydride (TpSA).    -   Hexadecenyl succinic anhydride (HDSA)    -   Octadecenyl succinic anhydride (ODSA)    -   Mixture (50:50) of ODSA and HDSA

Ethanol 96%, denaturated.

4 M HCl, and 4 M NaOH.

Na₂CO₃

Milli-Q® ultrapure water (Millipore).

Dialysis tubes of regenerated cellulose with a molecular weight cutoffof 12-14 kDa,

Spectr/Por™ (Spectrum Medical Industries).

Ultrafiltration membranes (MWCO 10 kDa and 3 kDa).

EXAMPLE 1 High-MW OSA-HA, Initial pH 9.0, Ethanol Precipitation

HA (batch MAF 145 SD, 1.42 g) was dissolved overnight at roomtemperature in Milli-Q water (200 mL) before adjusting pH to 9.0 with 4M NaOH. OSA (1 mL, 4.54 mmol) was added under strong agitation. Thesolution was left to react on strong agitation (approx 600 rpm) for 16hours at ambient temperature. 20 mL saturated NaHCO₃ was added to bufferthe reaction. After the reaction, the pH was adjusted to 6.8 with 1 MHCl. The product was recovered by ethanol precipitation by adding 96%ethanol (4 volumes) to give a final concentration of 80% v/v. Theprecipitate was recovered by centrifugation (3000 rpm, 15 min and 4°C.). The pellet was washed with 96% ethanol before re-dissolving in MQwater and freeze drying.

EXAMPLE 2 High-MW OSA-HA, Initial pH 11, Ethanol Precipitation

To each of three 50 mL solutions of Milli-Q water, HA (batch MAF 145 SD,1.13 g) was added and left to dissolve overnight at room temperature.The pH was adjusted to 11 with 4 M NaOH. Different amounts of OSA (1.10mL (5.23 mmol), 0.505 mL (2.62 mmol), 0.110 mL (0.52 mmol)) was added toeach of the three solutions under strong agitation. The solutions wereleft to react on strong agitation (approx 600 rpm) for 21 hours atambient temperature. All samples had a pH of around 4-5 after thereaction. The product was recovered by ethanol precipitation by adding96% ethanol (4 volumes) to give a final concentration of 80% v/v. Theprecipitate was recovered by centrifugation (3000 rpm, 15 min and 4°C.). The pellet was washed with 96% ethanol before re-dissolving in MQwater and freeze drying.

EXAMPLE 3 High-MW OSA-HA, Initial pH 9.0, pH Kept at 9-11, Dialysis

HA (batch MAF 145 SD, 0.75 g) was dissolved overnight at roomtemperature in Milli-Q water (150 mL) before adjusting pH to 9.0. OSA(1.42 mL, 6.25 mmol) was added under strong agitation. The solution wasleft to react on strong agitation (approx 600 rpm) for 16 hours atambient temperature. The pH was maintained around 9-11 by use of a pHstat (adding 1 M NaOH). The product was dialyzed 3×3 h against MQ water(4° C., 7 L, MWCO 12-14,000 Da), frozen and lyophilized.

EXAMPLE 4 High-MW OSA-HA, Initial pH 8.5, pH Kept at 9-11, Dialysis

HA (batch MAG 30014, 0.75 g) was dissolved overnight at room temperaturein Milli-Q water (150 mL) before adjusting pH to 8.5. OSA (1.42 mL, 6.25mmol) was added under strong agitation. The solution was left on strongagitation (approx 600 rpm) for 16 hours at ambient temperature. The pHwas maintained around 9-11 by use of a pH stat (adding 1 M NaOH). pH wasadjusted to 6.5 by use of 1 M HCl. The product was dialyzed 3×3 hagainst 0.2 M NaOH, and 3×3 h against MQ water (4° C., 7 L, MWCO12-14,000 Da), frozen and lyophilized.

EXAMPLE 5 Low-MW OSA-HA (30 and 100 kDa)

Low-MW HA (30 or 100 kDa, 2.5 g) was dissolved overnight at roomtemperature in Milli-Q water (50 mL) before adjusting pH to 8.5.Equimolar amounts of OSA (3.35 mL, HA:OSA ratio 1:1) or 1/10 of themolar concentration of HA (0.35 mL, HA:OSA ratio 10:1) was added understrong agitation. The solution was left to react on strong agitation(approx 600 rpm) for 16 hours at ambient temperature. The pH wasmaintained around 8.5-9.0 by use of a pH stat (adding 1 M or 0.5 MNaOH). The product was dialyzed 3×3 h against MQ water (4° C., 7 L, MWCO12-14,000 Da), frozen and lyophilized.

EXAMPLE 6 Product Characterization—Results and Discussion MolecularWeight

100 kDa OSA-HA from example 5 was analyzed using SEC-MALLS-VISC (mobilephase: 150 mM NaCl, 50 mM NaH₂PO₄, pH 7.0, 0.5 ml/min, injected volume:0.5 ml). Columns used: PL aquagel OH-40/OH-50/0H60. System: WatersAlliance HPLC system Waters 2410 RI detector and Wyatt MALLS detector.The data was processed using the ASTRA V software from Wyatt TechnologyCorp.

Degree of Substitution (DS)

DS was determined by ¹H NMR spectroscopy (10 mg/ml, D₂O, 80° C., 128scans, 400 MHz). The peaks from the OSA group were assigned by use of a2D-NMR (gCOSY).

Results and D

iscussion; High-MW HA During all experiments on high-MW HA theobservations have been the same: OSA forms amber-coloured oil drops thatgradually divide into smaller drops because of the agitation. At the endof the reaction, the solution was white and opaque like milk which canbe interpreted as formation of micelles or micro-scaleaggregates/droplets. Even after purification by precipitation ordialysis, where the excess of OSA is removed, this phenomenon was stillobserved to different extents.

During the first experiments on high-MW HA, precipitation in 80% ethanolwas used to remove the surplus/by-product of the OSA modification.However, due to problems with getting the product to precipitatecompletely, dialysis against Milli-Q water was chosen as a bettermethod.

The initial preparations of high-MW OSA-HA all showed changes insolution properties. One example is that they all stabilized foam veryefficiently for several hours; this was simply tested by shaking a 1%solution followed by visual inspection. Another observation during thefirst experiments was that the pH value of the solution declinesgradually during the reaction. Therefore, it was necessary to eitherbuffer the system, e.g., with NaCO₃ or by use of a pH stat. It isimportant that the pH value remains above 8.0 for the reaction toproceed, and below 9.0 to avoid removing the OSA groups by hydrolysis.

NMR spectroscopy was attempted on the high-MW OSA-HA products, butbecause of solubility problems only some very weak peaks of OSA and HAwere observed, and no DS could be determined. In all cases the yieldswere close or slightly higher than the amount of starting material HA(determined by weighing the lyophilized products).

Results and Discussion; Low-MW HA

Four separate experiments were performed and are summarised in Table 1together with DS from ¹H-NMR spectroscopy and yields. The DS iscalculated by comparing the intensity of the vinyl protons of OSA (5.4and 5.6 ppm) with that of the acetyl protons (2.0 ppm).

The ¹H NMR spectrum of the 30 kDa OSA-HA was elucidated by 2D NMRspectroscopy (gCOSY), and the partially assigned peaks are given in FIG.2, showing the ¹H NMR spectrum of the 100 kDa OSA-HA (14919-033).

Conclusively, in these experiments both high- and low-MW hyaluronic acidwas successfully modified with 2-octen-1-yl succinic anhydride (OSA).

TABLE 1 Results from the preparation of Low-MW OSA-HA. Batch 14658-12914658-131 14658-133 14919-033 14919-038 HA:OSA 1:1 1:1 10:1 1:1 10:1 MW30 kDa 30 kDa 30 kDa 100 kDa 100 kDa Yield 2.1 g 2.1 g 2.8 g 2.7 g 2.0 gDS 11.5% 12% 2.6% 18.8% 1.6%

EXAMPLE 7 Low-MW OSA-HA Derivatives (14 kDa)

Low-MW HA (14 kDa, 2.5 g) was dissolved at room temperature in Milli-Qwater (50 mL) before adjusting pH to 8.5. Equimolar amounts of OSA (3.35mL, HA:OSA ratio 1:1) or 1/10 of the molar concentration of HA (0.34 mL,HA:OSA ratio 10:1) was added under strong agitation. The solution wasleft to react on strong agitation (approx 600 rpm) for 16 hours atambient temperature. The pH was maintained around 8.5-9.0 by use of a pHstat (adding 0.5 M NaOH). The product was dialyzed 3×3 h against MQwater (4° C., 7 L, MWCO 12-14,000Da), frozen and lyophilized. Yieldsafter purification and freeze-drying were 2.1 g and 2.1 g, respectively.DS were determined as described in Example 6 to 11.5% and 2.6%,respectively.

EXAMPLE 8 Low-MW Phenyl-Succinic Anhydride (PhSA) HA Derivatives (14kDa)

Low-MW HA (14 kDa, 2.5 g) was dissolved at room temperature in Milli-Qwater (50 mL) before adjusting pH to 8.5. Equimolar amounts of PhSA (2.8g, HA:PhSA ratio 1:1) or 1/10 of the molar concentration of HA (0.28 g,HA:PhSA ratio 10:1) was added gradually under strong agitation. Thesolution was left to react on strong agitation (approx 600 rpm) for 16hours at ambient temperature. The pH was maintained around 8.5-9.0 byuse of a pH stat (adding 0.5 M NaOH). The product was dialyzed 3×3 hoursagainst MQ water (4° C., 7 L, MWCO 12-14,000Da), frozen and lyophilized.Yields after purification and freeze-drying were 2.5 g and 2.4 g,respectively. DS were determined as described in Example 6 to 15.1% and2.6%, respectively.

EXAMPLE 9 Low-MW 2-nonen-1-ylsuccinic anhydride (NSA) HA derivatives (14kDa)

Low-MW HA (14 kDa, 2.5 g) was dissolved at room temperature in Milli-Qwater (50 mL) before adjusting pH to 8.5. Equimolar amounts of NSA (3.55mL, HA:NSA ratio 1:1) or 1/10 of the molar concentration of HA (0.35 mL,HA:NSA ratio 10:1) was added under strong agitation. The solution wasleft to react on strong agitation (approx 600 rpm) for 16 hours atambient temperature. The pH was maintained around 8.5-9.0 by use of a pHstat (adding 0.5 M NaOH). The product was dialyzed 3×3 h against MQwater (4° C., 7 L, MWCO 12-14,000Da), frozen and lyophilized. Yieldsafter purification and freeze-drying were 2.4 g and 2.2 g, respectively.DS were determined as described in Example 6 to 11.4% and 2.1%,respectively.

EXAMPLE 10 Low-MW 2-dodecen-1-ylsuccinic anhydride (DSA) HA derivatives(14 kDa)

Low-MW HA (14 kDa, 2.5 g) was dissolved at room temperature in Milli-Qwater (50 mL) before adjusting pH to 8.5. Equimolar amounts of DSA (4.20mL, HA:DSA ratio 1:1) or 1/10 of the molar concentration of HA (0.42 mL,HA:DSA ratio 10:1) was added under strong agitation. The solution wasleft to react on strong agitation (approx 600 rpm) for 16 hours atambient temperature. The pH was maintained around 8.5-9.0 by use of a pHstat (adding 0.5 M NaOH). The product was dialyzed 3×3 h against MQwater (4° C., 7 L, MWCO 12-14,000Da), frozen and lyophilized. Yieldsafter purification and freeze-drying were 2.2 g and 2.2 g, respectively.DS were determined as described in Example 6 to 2.2% and 1.7%,respectively.

EXAMPLE 11 Low-MW tetrapropylsuccinic anhydride (TpSA) HA derivatives(14 kDa)

Low-MW HA (14 kDa, 2.5 g) was dissolved at room temperature in Milli-Qwater (50 mL) before adjusting pH to 8.5. Equimolar amounts of TpA (3.25mL, HA:TpSA ratio 1:1) or 1/10 of the molar concentration of HA (0.33mL, HA:TpSA ratio 10:1) was added under strong agitation. The solutionwas left to react on strong agitation (approximately 600 rpm) for 16hours at ambient temperature. The pH was maintained around 8.5-9.0 byuse of a pH stat (adding 0.5 M NaOH). The product was dialyzed 3×3 hagainst MQ water (4° C., 7 L, MWCO 12-14,000 Da), frozen andlyophilized.

EXAMPLE 12 Various Low-MW ASA HA Derivatives (14 kDa)

Eleven separate experiments were performed modifying LMW HA (14 kDa)with five different ASAs (see FIG. 3 for the ASA names, structures, andabbreviations) at two different HA:ASA molar ratios (1:1 and 10:1). Theresulting products were purifies by dialysis to remove excess reagentand byproducts. The degree of substitution (DS) was determined onmonomer basis by ¹H NMR spectroscopy. Yield was determinedgravimetrically the freeze dried samples. Molecular weight wasdetermined by SEC-MALLS-VISC. To avoid material getting stuck on the GPCcolumns the temperature was adjusted from 4° C. to 15° C. in theauto-injector. All results from the analyses are summarised in Table 2.

TABLE 2 Results from the preparation and characterisation of ASAmodified LMW HA (14 kDa starting material). Degree of MolecularConformational Ratio substitution Yield weight, plot factor; v SampleASA (ASA:HA) (%) (g) M_(w) (kDa) (R_(g) ~ M^(v)) 14658-142 OSA 1:1.259.7 2.70 17.0 0.35 ± 0.04 14658-144 OSA 1:0.125 3.5 2.37 14.2 0.56 ±0.05 14658-148 PhSA 1:1.25 1.8 2.19 13.8 0.62 ± 0.04 15286-017^(a) PhSA1:1.25 15 1.80 15.9 0.67 ± 0.02 14658-150 PhSA 1:0.125 3.4 2.19 14.30.58 ± 0.02 15286-010 NSA 1:1.25 11 2.35 21.1^(c) 0.04 ± 0.02 15286-012NSA 1:0.125 2.1 2.24 14.4 0.20 ± 0.01 15286-014^(b) DSA 1:1.25 2.2 2.2014.1 0.61 ± 0.02 15286-020^(b) DSA 1:0.125 1.7 2.24 14.0 0.49 ± 0.0215286-028 TpSA 1:1.25 * * * * 15286-037 TpSA 1:0.125 * * * *^(a)Repeated and downscaled version of 14658-148. PhSA was added insmall portions instead of all at once. ^(b)Problems with DSA—too thickto disperse efficiently with normal stirring; an oil phase was formedduring dialysis that had to be removed by pipette and discarded.^(c)Bimodal distribution; peak 1: 15.5 kDa, peak 2: 27.2 kDa *Currentlybeing analyzed.

As can be seen from the results summarized in table 2, thederivatization reaction runs smoother when a lower DS is the desiredoutcome. The obtained DS's are quite similar for all the different ASAs,except for the PhSA which apparently is very instable in water,resulting in a low DS value (2.19) for sample 14658-148. The experimentwas repeated where the PhSA powder was added gradually to theHA-solution. This resulted in a DS of 15% (15286-017), showing thatgradual addition of the ASA could be a way of increasing thesubstitution on HA.

For the LMW HA modified with DSA, the DS values are also quite low atthe higher ASA:HA ratio. This is probably because of the high viscosityof the DSA phase. In addition, the droplets of non-reacted ASA couldstill be seen after dialysis and freeze drying. This had to be removedmanually by a pipette.

Probably, the DS and purity of the DSA-HA can be improved by increasingthe temperature during the reaction combined with stronger agitation.Adding the DSA gradually may also increase the amount of DSA reactedwith HA. The TpSA samples (15286-037 and 15286-028) have not yet beenanalyzed yet.

By plotting radius of gyration (Rg) as against molecular weight (Mw) indouble logarithmic scale, one can obtain information about theconformation of the polymer. The relationship Rg and Mw is summarised inHaug's triangle (FIG. 4).

Conformational plots were made for all of the different ASA-HA, and theresults are given in Table 2. Most of the samples show factors similarto that of random coils, which is also the conformation of non-modifiedHA. The only exceptions, are the highly modified OSA-HA's, which have aconformation similar to that of a sphere, and the NSA-HA's that alsoshow very low conformational factors (0.20 and 0.04) indicatingaggregation or impaired column separation, perhaps because ofinteractions with the column material. Similarly, looking at theconcentration profile (RI-signal) from the SEC-MALLS-VISC analysis (SeeFIG. 5), there is an aggregation peak at an earlier elution time, atapproximately 35 minutes, for sample 15286-010 (11% NSA). Thisaggregation phenomenon is also indicated by the slight increase in theapparent MW (Table 2) for samples 14658-142 (9.7% OSA modified HA) and15286-010 (11% modified NSA-HA).

In conclusion, low MW hyaluronic acid (14 kDa) was successfully modifiedwith diverse aryl/alkyl succinic anhydrides. High DS products of OSA-HAand NSA-HA show some aggregation tendencies and changes in conformation,probably caused by hydrophobic interactions.

EXAMPLE 13 Surface Activity of OSA-HA (14 kDa) in Aqueous Solution

Solutions of 14 kDa OSA-HA (DS=9.7%, batch 14658-142) and unmodified LMWHA (30 kDa) were prepared in MQ-water according to the concentrationsgiven in Table 3. The samples were analyzed by surface tensionmeasurements using a surface tensiometer (Wilhelmy plate). Surfacetension of the solvent (water) was determined to 72 mN/m with the samemethod. Results of the surface tension measurements are given in FIG. 6and summarized in Table 3. As can be seen, the surface tension decreaseswith increasing concentration of OSA-HA. Comparing with the pure solvent(MQ-water) and LMW-HA (30 kDa), the surface tension is much lower forthe HA derivatives.

Further, it can be seen that the surface tension continues to decreasein a time dependant manner for the OSA-HAs. This can be explained by thefact that OSA-HA works as a high MW surfactant, using long time todiffuse to the surface of the solution (since diffusion speed isinversely proportional to MW). More surface active polymer at thesurface gives lower surface tension. This time dependence is also afurther proof of the OSA moieties actually being covalently bound to theHA, and not only co-existing with HA in the solution.

This further implies that OSA modification of HA is not rendering ithydrophobic, but amphiphilic. These properties can potentially beexploited in systems where lower surface tension is needed (e.g., localophthalmic) or where emulsifying properties are needed to stabilizeemulsions or foams in cosmetics or pharmaceutical formulations.

TABLE 3 Surface tension measurements of OSA-HA solutions in MQ-water at3200 seconds. LMW OSA-HA Concentration (%) (14 kDa, DS = 9.7%) LMW HA(30 kDa) 0.01 64 mN/m — 0.1 52 mN/m 64 mN/m 1.0 40 mN/m —

EXAMPLE 14 Preparation of High MW ASA Derivatives by High Shear Mixing

HA (4 g, MAG30021) was dissolved overnight in 400 mL MQ water. Solutionswere kept at room temperature (25° C.) or heated to 60° C. before Na₂CO₃(2 g) was added under shear (ULTRA-TURRAX 24 000 min⁻¹, 5 min). Then theASA was added according to reaction scheme presented in Table 4 andmixed under strong shear (ULTRA-TURRAX 24 000 min⁻¹, 5 min). Theresulting emulsions were left to react for 6 hours at the giventemperature (Table 4), then removed to room temperature over night. ThepH was adjusted to neutrality before the products were purified byultrafiltration (MWCO 10 000) until conductivity was below 15 μSi/cm.The products were frozen and lyophilized. NMR spectroscopy confirmedthat all the products were modified. Sample all samples gave turbidsolutions in 0.1 M NaCl at 1% w/v concentration.

TABLE 4 Reaction Scheme for preparation of high MW ASA-derivativesASA:HA Reaction temp. Sample ID ASA Carbon chain Molar ratio [° C.] 1OSA C8 1:1 25 2 OSA C8  1:10 25 3 ODSA C18 1:1 60 4 ODSA C18  1:10 60 5HDSA C16 1:1 60 6 DSA C12 1:1 60 7 DSA C12  1:10 60 ODSA: Octadecenylsuccinic anhydride, HDSA: Hexadecenyl succinic anhydride, DSA: Dodecenylsuccinic anhydride.

EXAMLE 15 ASA HA Stabilizes O/W Emulsions

The ASA-HAs no. 1, 3, 4, 5, 6, 7, and non-modified HA (MAG30014),prepared in example 14 were formulated with three cosmetic oils (mineraloil, diethylhexyl carbonate and ethylhexyl palmitate) according to thefollowing recipe:

-   -   1. 6 mL oil was added to 14 mL aqueous solution of 0.1 M NaCl        and 0.29% ASA-HA    -   2. The solution was mixed under strong shear for 25 seconds        (ULTRA-TURRAX at 24 000 min⁻¹).    -   3. The emulsions were left at room temperature in the dark for 8        weeks, being evaluated visually after 24 hour and 8 weeks for        stability.

All derivatives showed increased emulsion stability compared to thecontrol and the non-modified starting HA (see FIG. 7 for samples after24 hour and 8 weeks for ethylhexyl palmitate). This shows that ASA-HAcan be used as emulsifiers in cosmetics or advanced drug deliverysystems based on emulsions.

EXAMPLE 16 Various Low-MW ASA HA Derivatives with Long Alkyl Chains

Low MW ASA HA was prepared as described in example 15 for high MW HA,the only difference being that the starting concentrations of HA (23kDa) were 2% w/v. All samples in Table 5 were prepared at 60° C. andpurified by ultrafiltration (MWCO 3000 kDa) and lyophilization. The DSwere determined by ¹H NMR spectroscopy as described in example 6.

TABLE 5 Reaction scheme for preparation of low MW HA derivatives withlonger alkyl chains. DS Ratio (NMR Sample # ASA (HA/ASA) spectroscopy)Yield 15286-109-1 DSA 1:1.25   2.12% 4.09 g (HA: 23 kDa) 15286-109-2 DSA1:0.125 Confirmed 3.44 g (HA: 23 kDa) modified 15286-118-1 HDSA/ODSA1:1.25  13.14% 6.73 g (HA: 23 kDa) (50:50) 15286-118-2 HDSA/ODSA 1:0.125 1.06% 3.84 g (HA: 23 kDa) (50:50) 15286-120-1 ODSA 1:1.25  12.94% 6.15g (HA: 23 kDa) 15286-120-2 ODSA 1:0.125  0.08% 2.96 g (HA: 23 kDa)

EXAMPLE 17 Free Radical Scavenging Properties of Phenyl Succinic Acid

PhSA-HA (14 kDa) (10 mg/ml) in aqueous solution has been shown todegrade much faster than non-modified HA in the presence of hydroxylradicals (generated by Cu^(2+/)H₂O₂) followed by both streak cameraobservations and light scattering (DLS/SLS) studies. This shows thatPhSA-HA has a potential as a free-radical scavenging agent for potentialuse in cosmetic formulations.

EXAMPLE 18 Determination of Critical Aggregation Concentration (CAC) ofOctenyl Succinic Anhydride—Hyaluronic Acid Derivative (OSA-HA, DS 16%)

The critical aggregation concentration (CAC) of an OSA-HA derivative wasdetermined by calorimetry using an isothermal titration calorimeterVP-ITC (Microcal LLC, USA). A concentrated solution of OSA-HA (0.294 mL,15 mg/mL in distilled water) was used to titrate distilled water (1.4615mL) in the calorimeter sample cell. A solution of OSA-HA (2 pL, 15mg/mL) was injected every 300 seconds, and enthalpy variations (ΔH) inthe sample cell were recorded over time as shown in FIG. 8.

ΔH was plotted as a function of the OSA-HA concentration in the samplecell, and the CAC of OSA-HA was determined at the break of the curve.Each experiment was repeated three times and the CAC was provided as anaveraged value. For example, the CAC of OSA-HA with a degree ofsubstitution (DS) of 16% was 0.45 mg/mL (FIG. 9).

This study confirmed the existence of associative properties of OSA-HA.Moreover, it indicated the potential formulation of these derivativesinto micelles and/or micro-/nanoparticles, making them suitable for usein the encapsulation and delivery of hydrophobic compounds such ashydrophobic cosmetic bioactives and drugs.

EXAMPLE 19 Determination of Critical Aggregation Concentration (CAC) ofOctenyl Succinic Anhydride—Hyaluronic Acid Derivative (OSA-HA, DS 44%)

The CAC of OSA-HA with a degree of substitution of 44% was determined byfluorescence spectroscopy using a spectrofluorometer (FluoroMax, Spex,United States) thermostated with a water bath (Julabo F10, Merck, UnitedStates). Nile Red was employed as the fluorescent probe. Fluorescencewas measured on a range of OSA-HA solutions (Table 6) prepared indifferent phosphate buffers (Table 7).

TABLE 6 OSA-HA solutions Concentration Solution OSA-HA (mg/mL) 1 0.00012 0.0002 3 0.0006 4 0.001 5 0.002 6 0.006 7 0.01 8 0.02 9 0.06 10 0.1 110.2 12 0.6 13 1.0

TABLE 7 Phosphate buffers Concentration Concentration Buffer NaCl (M)NaH₂PO₄ (M) 1 0.15 0.01 2 0.50 0.01 3 1.00 0.01 4 1.50 0.01

Nile Red (3.184 mg) was dissolved in a mixture of THF and acetone(50/50, 10 mL). This solution (10 μL) was incubated with each OSA-HAsolution (10 mL) under stirring, overnight, in the dark and at roomtemperature. Each solution was analyzed at 25° C. at an excitationwavelength of 543 nm whereas emission spectra were recorded from 580 to700 nm. The excitation slit was set to 1 and the emission slit wasadjusted for each solution. The intensity of the fluorescence emission(I) was plotted as a function of the wavelength (λ).

The wavelength corresponding to the maximum intensity (λ max) wasdetermined by fitting the curve I vs. λ with a polynomial function oforder 6. Each λ max value was the average of three measurements.

In order to determine the CAC, λ max was plotted as a function of thepolymer concentration (C). The CAC was deduced at the inflexion point ofthe curve λ max vs. C (FIG. 10).

In FIG. 10 the CAC of OSA-HA (DS=44%) was somewhere between 0.003 and0.004 mg/mL. This phenomenon was not observed for unmodified HA. Indeedfluorescence could not be detected at any HA concentrations which meansthat it was not possible to solubilize Nile Red in HA solutions. Thisevidences the presence of polymeric assemblies in OSA-HA solutions.

EXAMPLE 20 Influence of the Salt Concentration on CAC of OSA-HA (DS=44%)

The same experimental set-up as the one described in the previousexample was used to study the influence of salt concentration on thevalue of CAC of OSA-HA (DS=44%). The results are shown in table 8 belowas well as in FIG. 11.

TABLE 8 Extend of Concentration of NaCl Onset of CAC the transition CAC(M) (mg/mL) (mg/mL) (mg/mL) 0.15 0.002  0.002-0.006 0.003-0.004 0.50.0006 0.0006-0.006 0.003 1.0 0.0006 0.0006-0.006 0.002 1.5 0.00020.0002-0.002 0.0006-0.0007

EXAMPLE 21 Zeta Potential of OSA-HA (DS=44%) Polymeric Micelles

The zeta potential of OSA-HA (DS=44%) polymeric micelles was determinedby capillary electrophoresis (Zetasizer 3000HS, Malvern, United Kingdom)coupled to a Doppler laser interferometer. Measurements were recorded at25° C. OSA-HA was dissolved in 10-3 M NaCl (at a concentration of 1mg/mL) prior to the measurement. The zeta potential of OSA-HA (DS=44%, 1mg/mL in 10-3 M NaCl) was evaluated to approximately −25 mV (FIG. 12).

EXAMPLE 22 Transmission Electron Microscopy of OSA-HA (DS=44%) PolymericMicelles

Microscopic observations of OSA-HA (DS=44%) polymeric micelles were madewith a transmission electron microscope (EM 410, Philips, TheNetherlands). Samples were deposited on ionised carbon coated coppergrids and stained with an aqueous uranyl acetate solution (2%).Microscopic snapshots clearly showed that the OSA-HA polymeric micellesare spherical in shape and have submicronic dimensions typically from 50to 200 nm (data not shown). This is shown in FIG. 13.

1. A process of producing a hyaluronic acid derivative or a salt thereofcomprising n repeating units of formula (I):

wherein (a) in at least one repeating unit, one or more of the R1, R2,R3, and R4 groups is an alkyl-/aryl-succinic acid of formula (II):

wherein at least one of the R5, R6, R7, and R8 groups is an alkyl- oraryl-group and the other R5, R6, R7, and R8 groups are hydrogen, and theoxygen labelled “ester” forms an ester bond with structure (I); and (b)in the other repeating units, the R1, R2, R3, and R4 groups are hydroxylgroups; the process comprising the steps of: (1) reacting hyaluronicacid (HA) or a salt thereof in an aqueous solution with one or morealkyl-/aryl-succinic anhydride (ASA) of formula (III):

wherein at least one of the R5, R6, R7, and R8 groups is an alkyl- oraryl-group and the other R5, R6, R7, and R8 groups are hydrogen; and (2)recovering the hyaluronic acid derivative.
 2. The process of claim 1,wherein one of the R1, R2, R3, and R4 groups is an alkyl-succinic acidof formula (II).
 3. The process of claim 1, wherein two of the R1, R2,R3, and R4 groups are an alkyl-succinic acid of formula (II).
 4. Theprocess of claim 1, wherein three of the R1, R2, R3, and R4 groups arean alkyl-succinic acid of formula (II).
 5. The process of claim 1,wherein at least one of the R1, R2, R3, and R4 groups is analkyl-succinic acid of formula (II), wherein the alkyl group is a C₁-C₂₀alkyl group.
 6. The process of claim 1, wherein at least one of the R1,R2, R3, and R4 groups is an alkyl-succinic acid of formula (II), whereinthe alkyl group is a propyl, 2-octenyl, 2-dodecenyl, 2-hexadecenyl, or2-octadecenyl group.
 7. The process of claim 1, wherein one of the R1,R2, R3, and R4 groups is an aryl-succinic acid of formula (II).
 8. Theprocess of claim 1, wherein two of the R1, R2, R3, and R4 groups are anaryl-succinic acid of formula (II).
 9. The process of claim 1, whereinthree of the R1, R2, R3, and R4 groups are an aryl-succinic acid offormula (II).
 10. The process of claim 1, wherein at least one of theR1, R2, R3, and R4 groups is an aryl-succinic acid of formula (II),wherein the aryl-group is phenyl.
 11. The process of claim 1, whereinthe hyaluronic acid derivative or salt thereof has an average molecularweight of between 10,000 and 1,500,000 Da.
 12. The process of claim 1,wherein the hyaluronic acid derivative or salt thereof has an averagemolecular weight of between 20,000 and 50,000 Da.
 13. The process ofclaim 1, wherein the hyaluronic acid derivative or salt thereof has anaverage molecular weight of between 50,000 and 500,000 Da.
 14. Theprocess of claim 1, wherein the hyaluronic acid derivative or saltthereof has an average molecular weight of between 80,000 and 300,000Da.
 15. The process of claim 1, wherein the hyaluronic acid derivativeor salt thereof has a Degree of Substitution (DS) in the range of 1-90%.16. The process of claim 1, wherein the hyaluronic acid derivative orsalt thereof has a Degree of Substitution (DS) in the range of 10-50%.17. The process of claim 1, wherein the hyaluronic acid derivative orsalt thereof has a Degree of Substitution (DS) in the range of 14-40%.18. The process of claim 1, wherein the hyaluronic acid derivative orsalt thereof has a Degree of Substitution (DS) in the range of 18-25%.19. The process of claim 1, further comprising recombinantly producingthe hyaluronic acid (HA) or salt thereof.
 20. The process of claim 19,wherein the hyaluronic acid (HA) or salt thereof is recombinantlyproduced in a Bacillus host.